O. García-Beltrán et al. / European Journal of Medicinal Chemistry 67 (2013) 60e63
61
The novel fluorescence probe was prepared via a conventional
two-step synthesis from commercial precursors. 4-Diethylamino
salicylaldehyde (1) was condensed with ethyl malonate (Kno-
evenagel) giving ethyl 7-diethylamino-2-oxo-2H-chromene-3-
carboxylate (2). This coumarin was condensed with 2-amino-2-
(hydroxymethyl)propane-1,3-diol (TRIS) to afford AGD, by analogy
with a literature procedure (Scheme 1). The 1H NMR and 13C
NMR spectra of AGD are available in the Supporting Information
(Fig. 1A and B).
The absorption and emission spectral studies were performed in
pH 7.4 HEPES buffer at room temperature. AGD shows an absorp-
tion maximum at 430 nm (Fig. 3; Supporting Information), a molar
extinction coefficient (
480 nm (Fig. 4; Supporting Information). The quantum yield of this
probe is 0.103 and its Stokes shift is 2423 cmꢀ1 Fe2þ, Fe3þ, Ca2þ
Co2þ, Mg2þ, Mn2þ, Zn2þ, Cd2þ, Pb2þ and Hg2þ chlorides were added
3
) of 5245 Mꢀ1 cmꢀ1 and an emission band at
,
at a final concentration of 200 mM to a 20 mM solution of AGD. The
effects of all but Fe2þ and Fe3þ were negligible, and the fluorescence
quenching by Fe2þ was about twice as intense as that of Fe3þ (Fig.1).
With excitation at 430 nm, the fluorescence emission intensity of
AGD at 480 nm decreases by about one third upon increasing the
Fig. 1. Fluorescence spectra of AGD (20
m
M) in the presence of different metal cations
(200 M) in 20 mM HEPES buffer, pH 7.4.
m
concentration of Fe2þ from 0 to 200
mM (Fig. 2) and the quantum
yield decreases to 0.0057. A good linear relationship between
fluorescence intensity and Fe2þ concentration was observed, with a
correlation coefficient as high as 0.99097 (Inset to Fig. 2).
The stability constant Ka of the AGD-Fe2þ interactions was
determined using the BenesieHildebrand equation, which gives a
value of 4.19 ꢁ 104 Mꢀ1 (Fig. 5; Supporting data). A literature report
suggests that AGD-Fe2þ complexes might be polymeric, considering
the steric effects, where an iron atom coordinates with the three
hydroxyls of one AGD molecule and the two carbonyls of the
following molecule (Fig. 6; Supporting data) [7]. The ESI spectrum
exhibited a peak at m/z 420.3426 assignable to [AGD þ Fe(II)]þ (Fig. 7;
Supporting data), indicative of a 1:1 stoichiometry, but it does not
rule out the possibility of labile polymeric complexes. However,
although the hydroxyl groups of the 2-amino-2-(hydroxymethyl)
propane-1,3-diol, the carbonyl groups of the a-pyrone and amide,
might all bind the iron atom in the monomeric species detected, the
conformation adopted to form such a complex is unclear and should
give rise to further work in coordination chemistry.
To assess the practical applicability of AGD as a Fe2þ selective
fluorescent sensor, we carried out competitive experiments with
20
Fe3þ, Zn2þ, Cu2þ, Co2þ, Ca2þ, Mn2þ, Mg2þ, Pb2þ, Cd2þ, and Hg2þ
after which 200
M Fe2þ was added. As shown in Fig. 3, these ex-
periments demonstrate that none of the selected ions interfere to
any obvious extent with the detection of Fe2þ
Fluorescent probes available for iron are few, and many of them
cannot be used for biological applications. Moreover, bifunctional
iron-chelating compounds with preference for a specific organelle
are scarce, since the vast majority are found in the cytoplasm.
Consequently, the behavior of AGD was studied in SH-SY5Y human
neuroblastoma cells loaded with Fe3þ by incubating them for 24 h
with ferric nitrilotriacetic acid complex (FeNTA), monitoring
changes in fluorescence with a microplate fluorescence reader.
Reversibility is another important feature of excellent chemical
mM AGD pretreated with 200 mM of each of the following ions:
Fig. 2. Fluorescence spectra of AGD (20
pH 7.4 HEPES buffer. Inset shows the relationship between fluorescence intensity and
Fe2þ concentration (10e100
M).
m
M) with different concentrations of Fe2þ in
,
m
m
.
probes. Therefore, experiments were conducted adding bipyridyl (a
molecule with high affinity for the metal) to examine the revers-
ibility of the AGD-Fe2þ interaction (Fig. 4A). These showed that the
fluorescence intensity of Feeloaded cells treated with AGD (Fig. 4B)
increased with the addition of bypiridyl (BIP, Fig. 4C). Subsequent
addition of ferrous ammonium sulfate (FAS) quenched the fluo-
rescence again (Fig. 4D). This experiment was also followed by
epifluorescence microscopy, visualizing the change in fluorescence
intensity caused by the different treatments. The mitochondria
were labeled by incubation with Mito-Tracker (Fig. 5A). The
OH
O
O
O
OH
O
N
H
O
a
b
OH
N
OH
N
O
N
O
O
AGD
2
1
Scheme 1. Synthetic route to AGD. Reagents and conditions: a) diethyl malonate, piperidine, acetic acid, EtOH, reflux 6 h; b) 2-amino-2-hydroxymethylpropane-1,3-diol, EtOH, reflux 48 h.
Garcia-Beltran, Olimpo
